Journal of Applied Pharmaceutical Science Vol. 7 (09), pp.

122-133, September, 2017

Available online at http://www.japsonline.com
DOI: 10.7324/JAPS.2017.70917
ISSN 2231-3354

Predictive mathematical modeling for EC50 calculation of antioxidant

activity and antibacterial ability of Thai bee products

Rungsiri Suriyatem1, Rafael A. Auras2, Pilairuk Intipunya1, Pornchai Rachtanapun3*

1
Division of Food Science and Technology, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai 50100, Thailand.
2
School of Packaging, Michigan State University, East Lansing, Michigan 48824, USA.
3
Division of Packaging Technology, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai 50100, Thailand.

ARTICLE INFO ABSTRACT

Article history:
Received on: 12/06/2017
Accepted on: 24/07/2017
Available online: 30/09/2017
Key words:
EC50 prediction, honey,
phenolic compounds, pollen,
propolis.
Antioxidant activities of bee products from Thailand (honey, bee pollen and propolis) via the 2,2-diphenyl-1-
picrylhydrazyl (DPPH) and 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulphonate)(ABTS) assays were
determined. The prediction of the EC50 (the half maximal effective concentration) were studied using the
logistic, sigmoidal, dose response, and asymmetric 5 parameters (5P) regression models. The antimicrobial
ability was tested against Staphylococcus aureus (TISTR 517), Bacillus cereus (TISTR 687), and Escherichia
coli(TISTR 1261). Propolis extract with higher total phenolic content (TPC) exhibited more effective antiradical
action against the DPPH and ABTS, followed by bee pollen extract and honey. All four regression models could
be used to estimate the EC50 of the bee products. However, the dose-respond and 5P provide the better EC50
prediction for the bee products than the others based on the comparability of their results to those of right-angled
triangle method. Thai bee products had effective antimicrobial activities on each test microorganism. The
antimicrobial potency of the bee products was ranged in the order: propolis> bee pollen >honey. Results
revealed that antioxidant activity and antimicrobial ability of the bee products correlated with the TPC values.

INTRODUCTION
Anti-oxidative action is one of the physiological
functions of many compounds found in foods (Nagai et al.,
2001). This action is assumed to protect living organisms from
oxidation, resulting in the prevention of various diseases such as
cancer and diabetes (Nagai et al., 2001). The antimicrobial
activity of chemical compounds, including antibacterial,
antifungal and antiviral activity, is important against infections
incited by microorganisms (Bogdanov, 2011). Plant polyphenols
are potential natural alternatives to synthetic antioxidant and
antimicrobial compounds (Siripatrawan et al., 2013). Bee
products, one of the essential sources of polyphenols, are
well known in traditional medicine dating back to ancient times.
* Corresponding Author
Pornchai Rachtanapun, Division of Packaging Technology, Faculty of
Agro-Industry, Chiang Mai University, Chiang Mai 50100, Thailand.
P: +66635492556, E-Mail: pornchai.r @ cmu.ac.th
© 2017 Rungsiri Suriyatem et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-
ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/).
Man utilized bee products in many ways, and now a days their
applications have expanded from healthy foods to medicinal
products. Bee products such as honey, bee pollen, royal jelly and
propolis from various geographical locations around the world
have been found to possess antioxidant and antimicrobial activities
(Buratti et al., 2007; Choi et al., 2006; Graikou et al., 2011).
Honey the nectar that the honey bees collect and process from
many plants(Ferreira Isabel et al., 2009)has been used in food as a
sweetening agent (Nagai et al., 2001) and food preservative since
ancient times (Ferreira Isabel et al., 2009; Nagai et al., 2001).
Honey normally consists of more than 150 substances, including
complex mixture of sugars and small amount of polyphenolic
compounds such as flavonoids and cinnamic acid derivatives
(Buratti et al., 2007; Ferreira Isabel et al., 2009). Bee pollen is a
fine, powder-like material produced by flowering plants and
collected by worker honey bees formed into granules with added
honey or nectar (Bogdanov, 2011).Bee pollen contains lipids,
proteins, sugars, amino acids, vitamins, carotenoids and poly-
phenolics (Graikou et al., 2011).
 Suriyatem et al. / Journal of Applied Pharmaceutical Science 7 (09); 2017: 122-133
Bee pollen is considered to be a nutrient-rich perfect food
and is commercially promoted as a dietary supplement. Propolis is
a sticky substance derived from plant resins collected by
honeybees (Bogdanov, 2011).
Propolis contains more than 300 constituents such as
polyphenols, sesquiterpene quinones, coumarins, steroids, amino
acids, and inorganic compounds (Choi et al., 2006; Siripatrawan et
al., 2013).The composition of propolis varies depending on the
season and on the botanical origin from which the plant resins
have been collected (Bosio et al., 2000). Propolisis now
recognized to have a wide range of biological activities, such as
antibacterial, anti-inflammatory, antioxidative, hepatoprotective,
and tumoricidal activities (Bosio et al., 2000; Miorin et al., 2003).
Determination of the antioxidant power of the bee
products involved the use of different methods such as the DPPH
(2,2- diphenyl-1-picrylhydrazyl), ABTS [2,2´-Azino-bis(3-
ethylbenzthiazoline-6-sulphonate)], TBARS (thiobarbituric acid
reactive substances) and β-carotene bleaching assays (Buratti et
al., 2007; Ferreira Isabel et al., 2009; Lachman et al., 2010;
Siripatrawan et al., 2013). The DPPH and ABTS assays have been
widely used to determine antioxidant activity of various plants and
other materials since they are stable free radicals and the
determination is simple. The half maximal effective concentration
(EC50), the concentration of antioxidant that causes a 50% decrease
in the radical absorbance, is commonly expressed by measuring
antioxidant results. In 1999, Alexader et al. (1999) presented a
simple and accurate mathematical method for calculation of the
EC50 called right-angle triangle. They suggested that the right-
angle triangle method is simple, accurate, and non-computational
technique for the calculation of the EC50 is needed. Now days, a
number of methods and software have been developed which
contain the functions for non-linear curve-fitting of the
experimental data and estimation of the EC50 value, making this
determination fast and particularly useful for laboratory test. Chen
et al. (2013) studied the EC50 estimation of antioxidant standards
(quercetin, catechin, ascorbic acid, caffeic acid, chlorogenic acid
and acetylcysteine) with DPPH assay using various computer
programs and mathematical models. All the statistical programs
they used provided similar EC50 values, however, the asymmetric
five-parameter equation in the GraphPad Prism software was
found to point out a best fit for their experiment. Recently, the
estimation of EC50 values for fungi with different methods using
computer programs were also reported by Li et al.(2015).Results
showed that among all the statistical programs they used, IBM
SPSS, GraphPad Prism, DPS were appropriated for EC 50
calculations of their samples.
To the best of our knowledge, there has been no research
that publishes the EC50 prediction of antioxidant activity for honey,
bee pollen and propolis using comparative different regression
models.
Thus, the objectives of the present work are: (1) to
evaluate the antioxidant activity of the extracts of propolis, bee
pollen and honey from Thailand, and (2) to identify the best model
for the prediction of EC50 from experimental data obtained via
123
DPPH and ABTS assays. The in vitro antimicrobial activity was
also investigated and it is reported.
EXPERIMENTAL
Materials
Longan honey and bee pollen were purchased from
Chiang Mai Healthy Product (Chiang Mai, Thailand). Dried
propolis extract selected from Chiang Mai province were
purchased from T. Man Pharma Co., Ltd. (Bangkok, Thailand).
Gallic acid, DPPH (2,2-Diphenyl-1-picrylhydrazyl), ABTS [2,2´-
Azino-bis(3-ethylbenzthiazoline-6-sulphonate)], Trolox (6-
hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid),2.0 N
Folin-Ciocalteu reagent and α-tocopherol were purchased from
Sigma-Aldrich (MO, USA); sodium chloride, methanol and
ethanol were purchased from QRёC (Auckland, New Zealand). L-
ascorbic acid, sodium carbonate and potassium persulfate were
purchased from Ajax Finechem Pty Ltd (New South Wales,
Australia); NB (nutrient broth) and NA (nutrient agar) were
purchased from HiMedia Laboratories Pvt. Ltd (Mumbai, India).
All reagents were of analytical grade and used as received.
Preparation of bee pollen extracts
Bee pollen extract was prepared according to the
procedure described by Morais et al. (2011). Bee pollen was
soaked in methanol at pollen-to-methanol ratio of 1:2 (w/v). The
mixture was left to macerate for 72 h at room temperature and
shaken by hand for 5 min twice a day. The pollen extract was
filtered through a Whatman filter paper No. 4 using a Buchner
funnel. The methanol extract was evaporated in a vacuum
evaporator (Thailand) and stored in an amber glass bottle at 4ºC
for further analysis.
Determination of total phenol content
The total phenol content (TPC) of the bee product
samples was determined using the Folin–Ciocalteau method as
described by Ahn et al. (2004) with slight modifications. The
sample (0.3 mL) was put in a test tube, and 3 mL of distilled
water, 0.25 mL of 2.0 N Folin-Ciocalteu reagent and 2.5 mL of 7%
(w/v) sodium carbonate were added. Each tube was covered with a
cap and shaken with a vortex mixer (Dragon Lab, China). After 30
min of incubation in a dark place at 25˚C, the absorbance was
measured at 760 nm with a spectrophotometer (Labomed, USA)
and compared to a calibration curve of gallic acid. The results are
presented as means of triplicate analyses and expressed in mg
gallic acid equivalents/g of sample (mg GAE/g sample).
Determination of antioxidant activity
The DPPH (2,2- diphenyl-1-picrylhydrazyl) scavenging
capacity of the bee products was monitored according to the
method described by Brand-Williams et al. (1995). A different
dilution of the samples (0.3 mL) was mixed with 0.06 mM DPPH-
methanolic solution (2.7 mL). The mixture was placed in a dark
room for 30 min. The absorbance at 516nm (A) was determined
124 Suriyatem et al. / Journal of Applied Pharmaceutical Science 7 (09); 2017: 122-133
with a spectrophotometer (Labomed, USA). This activity was
given as % DPPH scavenging and calculated using equation 1:
where Acontrol is absorption of DPPH solution, and Asample is
absorbance of the test sample. The half maximal effective
concentration (EC50)is the amount of sample necessary to decrease
the absorbance of DPPH by 50%. It was calculated by
interpolation from the graph of inhibition percentage against
sample concentration using a simple mathematical method based
on the principle of right-angled triangle(Alexander et al.,
1999).Ascorbic acid and α-tocopherol were used as positive
controls. All the analyses were carried out in triplicate.
Determination of Trolox equivalent antioxidant capacity
(TEAC)
For the TEAC assay, the procedure followed the method
described by Re et al.(1999). The TEAC assay is based on the
scavenging of the 2, 2’-azinobis-(3-ethylbenzothiazoline-6-
sulphonate) (ABTS) radical (ABTS•+). ABTS•+ was produced by
reacting 7 mM ABTS solution with 2.45 mM potassium persulfate
solution (1:1) (v/v) and storing it in the dark at room temperature
for 16 h before use. The ABTS•+ solution (1 mL) was diluted to
get an absorbance of 0.700±0.025 at 734 nm with methanol (40
mL). Bee product sample (0.3 mL) was added to the ABTS•+
solution (2.7 mL) and the absorbency was measured after 6 min.
The %inhibition of the sample was calculated using the formula
mentioned in the DPPH assay. The result was then compared with
a standard curve made from the corresponding readings of Trolox
(0–0.2 mM). Results were expressed as mg trolox equivalents/g
dried sample (mg TE/g sample).
EC50 prediction using statistical models
Data analysis for the free radical scavenging activity of
bee product samples was performed using the logistic, Boltzmann
(sigmoidal), log (agonist) vs. normalized responsevariable slope
(doseresponse), and asymmetric sigmoidal (five parameter, 5P)
mathematical models indicated in equations 2 to 5, respectively,
using JMP 10 (SAS Institute Inc., Cary, NC, USA) and SciDAVis
(version 2, Boston, MA).
Logistic
Boltzman (sigmoidal)
Doseresponse
Asymmetric sigmoidal
where x is log of concentration, y is response, y’ is normalized
response (0 to 100%), A1 is the baseline, A2is the maximum
response, x0 is center or logEC50, p is power, dx is time constant,
Hillslope is the steepness of the curve which has no units and s is
the symmetry parameter, which is unit less and xb is concentration
at the inflection point. For asymmetric sigmoidal model, the EC50
can be calculated from the xb, Hillslope and s parameters by using
equations 6 as followed:
Antimicrobial ability
Preparation of inoculums
Gram-positive (Staphylococcus aureusTISTR517 and
Bacillus cereus TISTR687) and Gram-negative (Escherichia coli
TISTR1261) organisms were obtained from the Division of
Biotechnology, Faculty of Agro-Industry, Chiang Mai University
(Chiang Mai, Thailand). S. aureus, B. cereus and E. coli were
cultured in NB at 30˚Cfor 24 h. The optical density (OD) of the
bacteria was adjusted to the standard of McFarland No.
0.5(Hindler et al., 1992)with 0.85 g sodium chloride/100 mL
sterile solution to achieve a concentration of approximately 10 8
CFU/mL. The final concentration of the cell numbers was
approximately 105-106 CFU/mL obtained by diluting 100 times
with sterile sodium chloride solution.
Determination of minimum inhibitory concentrations (MIC) and
minimal bactericidal concentration (MBC)
The MIC of the bee product samples was determined
using a broth dilution assay according to the procedure described
by Mazzola et al. (2009). One mL of NB medium was dispensed
in each of the 12 numbered test tubes (16 mm x 150 mm), except
for tube # 1. The tubes were autoclaved (IWAKI, Japan) at 121ºC.
For tubes#1 and # 2, 1 mL of test sample was introduced; tube # 2
was stirred and 1 mL was withdrawn and transferred to tube #3.
This serial dilution was repeated for all tubes up to tube # 11. Then
1 mL was removed from tube # 11.One mL of each test
microorganism was added to each tube. All tubes were incubated
at 30ºC for 24 h and the results were evaluated. The MIC of
bacteria was defined as the lowest concentration at which no
growth occurred. Tube # 12 is the positive control (NB +
inoculation).
The MBC test determines the lowest concentration at
which an antimicrobial agent will kill a particular
microorganism. The MBC is defined using a series of steps,
undertaken after the MIC test has been completed. The dilution
representing the MIC and at least two more concentrated test
sample dilutions was touched with a loop and streaked on a NA
plate and incubated at 30 ºC for 24 h. The MBC was determined
as the lowest concentration at which no growth appeared
(Taemchuay et al., 2009). The plates with streaking of each
inoculation were used as the control.
 Suriyatem et al. / Journal of Applied Pharmaceutical Science 7 (09); 2017: 122-133
Table 1: Total phenol content and antioxidant activity determined by DPPH and ABTS assays of bee product samples.
Sample Total phenol content
(mg GAE/g sample)
α-tocopherol (positive control) - 0.055 ± 0.001 a 0.032 ± 0.002 a
ascorbic acid (positive control) - 0.023 ± 0.002 b 0.023 ± 0.002 b
honey 0.57 ± 0.01 a
bee pollen 24.22 ± 0.34 b
propolis 237.18 ± 6.76 c
Note: Values in a column with the same letter are not significantly different (P≤0.05).
Statistical analysis
The data were analyzed by a one-way analysis of
variance (ANOVA) and Tukey HSD’s multiple range test (p≤0.05)
using the SPSS software (Version 11, SPSS Inc., Chicago, IL).
RESULTS AND DISCUSSION
Total phenol content
Thai honey, bee pollen and propolis were analyzed by the
proposed procedure and the results were expressed as mg GAE/g.
Polyphenols were found in our bee products. The values of their
total phenol content are shown in Table 1, and they are in the
following decreasing order: propolis extract > pollen extract >
honey. Buratti et al.(2007) also found a similar trend for bee
products. The TPC of our propolis (237.18 mg GAE/g sample)
agreed with values obtained by Ahn et al. (2004) in Korean
propolis (85-283 mg GAE/g) and by Moreira et al.(2008)in
Portuguese propolis (151-329 mg GAE/g). However, the TPC of
our propolis was higher than the values reported by Siripatrawan et
al.(2013) and Kumazawa et al.(2004)in Thai propolis,22.8-77.5
mg GAE/g and 31.2 mg GAE/g, respectively, and Choi et al.
(2006) in Brazilian propolis (120 mg GAE/g). On the other hand,
the TPC of Chinese propolis showed slightly higher values as
ranged from 262 to 299 mg GAE/g (Kumazawa et al., 2004).
Meanwhile, the TPC of our bee pollen extracts (24.22 mg GAE/g)
was in agreement with those of bee pollen reported from Portugal
(25.3-28.8 mg GAE/g) and Spain (18.6-32.2 mg GAE/g) (Pascoal
et al., 2014).The TPC of our honey (0.57 mg GAE/g)was
comparable to honey collected in Thailand (0.23-0.73 mg GAE/g)
by Jantakee and Tragoolpua (2015) and in Portugal (0.23-0.73 mg
GAE/g) by Ferreira et al. (2009). However, this value was lower
when compared to honey from Brazil (1.05 mg GAE/g) (Sant'ana
et al., 2014).The variation of TPC from the bee products from
various origins could be attributed to climate and environmental
factors such as humidity, temperature and soil composition.
Antioxidant activity
Antioxidants from natural sources are attractive
alternatives to synthetic antioxidants. Antioxidants can be used to
prevent diseases and oxidation of food products (Morais et al.,
2011). According to the complex nature of natural antioxidants,
Sakanaka and Ishihara (2008) suggested that the use of at least two
methods is recommended to evaluate and compare the antioxidant
capacity of a sample. In this research, we used the procedure based
125
DPPH assay ABTS assay
EC50 (mg/mL) EC50 (mg/mL) TEAC (mg TE/g sample)
768.4 ± 44.55 a
1053 ± 101.2 b
276.7 ± 6.117 c 92.29 ± 5.638 c 0.263 ± 0.016 c
1.499 ± 0.015 d 0.560 ± 0.039 d 43.35 ± 3.152 d
0.150 ± 0.004 e 0.054 ± 0.008 e 456.7 ± 69.90 e
on the reduction of DPPH and ABTS, stable free radicals, to
investigate the free radical-scavenging activity of the bee products.
The DPPH and ABTS assay has been widely employed to
determine the free radical scavenging ability of a variety of natural
antioxidants (Chen et al., 2013; Lachman et al., 2010;
Siripatrawan et al., 2013).The underlying mechanisms of
determining the activity using DPPH and ABTS can be
represented as Reaction 1 and 2, respectively(Boligon et al.,
2014):
(1)
(2)
In the DPPH assay, the purple DPPH• is reduced by
hydrogen–donating of antioxidant to the pale yellow DPPH–H. In
the ABTS scavenging process, first, ABTS•+ is generated by
reacting a strong oxidizing agent, potassium persulfate, with
ABTS salt. The blue–green ABTS•+ is converted back to its
colorless ABTS by hydrogen–donating of antioxidant.
The free radical scavenging activity of the methanolic
fraction of the honey, pollen extract and propolis extract was
measured at various sample concentrations by the DPPH assay and
ABTS assay and expressed as the EC50values in Table 1.Ascorbic
acid and -tocopherol, well known natural antioxidant, were used
as standards. The EC50 values calculated from DPPH and ABTS
assays for the bee products ranged between0.159–286.8 mg/mL
and 0.059–93.19 mg/mL, respectively. The average antioxidant
activities determined by the ABTS assay were two to three times
lower compared to values determined by the DPPH assay
(Lachman et al., 2010).This may probably because DPPH may
have limitation and show lower sensitivity to the bee products than
ABTS.ABTS•+ is applicable to both hydrophilic and lipophilic
antioxidants due to its solubility in both aqueous and organic
solvents while DPPH• is useable to hydrophobic systems since it is
dissolvable in organic media(Floegel et al., 2011). Therefore, the
ABTS method is reactive towards most antioxidants; whereas
some compounds react very rapidly by the DPPH assay.
The lower EC50 value indicates a higher antioxidant
activity for the product. The antioxidant activity values determined
by these two different assays (Table 1) – revealed that among the
bee products propolis had the stronger antioxidant power when
compared to bee pollen and honey but lower than the standards.
This outcome may be attributed to the large concentration of
126 Suriyatem et al. / Journal of Applied Pharmaceutical Science 7 (09); 2017: 122-133
phenolic compounds available in propolis. These results are in
.
accordance with results provided by Buratti et al.(2007) and Nagai
et al. (2001). The data collected by Buratti et al. (2007) via DPPH
assay showed that within the Italian bee products, propolis (IC50 =
1.0–2.1 mg/mL) had the highest antioxidant capacity followed by
royal jelly (IC50 = 1.4–2.3 mg/mL) and honey (IC50 = 5.0–15.5
mg/mL). Nagai et al. (2001) studied the anti-oxidative effects of
some honeys, royal jelly, and propolis from Japan using a lipid
peroxidation model. They discovered that the superoxide
scavenging activities of the bee product decreased in the following
order: propolis> royal jelly > honey.
Interestingly, the extracts of the bee products, which
exhibited higher activity, were those that contain a high phenol
level. Propolis was obviously most active among all the bee
product samples. The antioxidant activity seemed to be related to
the total phenol content of the extract. Similar phenomena have
been reported for propolis from Korea (Choi et al., 2006), Italian
bee products (Buratti et al., 2007), Czech honey (Lachman et al.,
2010), Brazilian honey (Sant'ana et al., 2014), Portuguese bee
pollen (Morais et al., 2011), Greek bee pollen (Graikou et al.,
2011), and Thai propolis (Siripatrawan et al., 2013). Flavonoids
and phenolic components played an important role in the free
radical scavenging capacity of the extract (Graikou et al., 2011).
Likewise, the different origins of the extracts may provide
different types and contents of the phenolic compounds in
propolis. Rutin, quercetin and naringenin were found to be the
main phenolic compounds in propolis collected from Nan
province, Thailand (Siripatrawan et al., 2013). Kumazawa et al.
(2004), who study the antioxidant activity of propolis of various
geographic origins, found that propolis contained antioxidative
compounds such as kaempferol and phenethylcaffeate showing the
strong antioxidant activity. Phenols exhibits an excellent property
of reducing spontaneous autoxidation of organic molecules
(Ingold, 1961) through a general class of mechanism called chain-
breaking. Chain breaking antioxidants operate by neutralizing
peroxide radicals to stop chain propagation of the radicals. To
inhibit the oxidation, an H atom from the phenols is transferred to
the oxidative chain carrying peroxyl radicals (ROO•)as
exemplified in Reaction 3(Foti, 2007):
Phenoxyl radical (Phenol-O•), generated as a product, is normally
nonreactive to oxygen (O2) and substrates (RH) (Reaction 4 and
5). This reduces the rate of the oxidation reaction(Ingold, 1961).
The Phenol-O• is then degenerated via the bimolecular self-
reaction or the reaction by another ROO• radical (Reaction 6 and
7).
(4)
(5)
(6)
(7)
Figure 1 shows the correlation between the total phenol and
antioxidant activity with 1/EC50 values measured from DPPH
assay of the tested bee products. Correlations of some natural
products from previous studies (Barreira et al., 2008; Harzallah et
al., 2016; PhomkaivonAreekul, 2009) are also showed (Figure 1).
The antioxidant activity of the products correlated with the total
phenolic contents. Ferreira et al.(2009)have tested honey form
Northeast Portugal and found that the higher antioxidant contents
and the lower EC50 values for antioxidant activity were obtained in
the darker honey which contained higher total phenolics. In the
study of Moreira et al.(2008), propolis from northeast and center
of Portugal was analyzed. Lower values of EC50 on DPPH
scavenging assay were obtained for northeast of Portugal, which
could be related with the higher total phenols content. However,
the strong relation between the phenolic compounds and
antioxidant activity was not found for the bee pollen studied by
Pascoal et al. (2014) and Morais et al. (2011), and they did not
give any reason.Previous studies have presented the effect of
phenolic compounds on antioxidant activity of other natural
products. He et al.(2015) studied antioxidant activity of
Pyruspashia flowers in China, and they found that antioxidant
effect of P.pashia flowers was related with phenolics content.
Barreira et al. (2008) determined an antioxidant activity and
polyphenols content of the extracts from various part of chestnut
such as flowers, leaves and fruits. They found that chestnut
flowers and leaves presented very good antioxidant activity while
chestnut fruits revealed the highest EC50 values. Their obtained
results are in agreement with the phenol contents determined for
each sample. In this work, we also show a correlation of the EC50
value with the total phenolic contents.
Fig. 1: Total phenolic (mg GAE/g) vs. 1/EC50 (mL/mg) of bee products and
some natural products. EC50 was obtained from DPPH assay.
EC50prediction using statistical models
EC50 is an important parameter to evaluate the
antioxidant activity of materials and it could be used to compare
the antioxidant capacity of various materials. The EC50 could be
 Suriyatem et al. / Journal of Applied Pharmaceutical Science 7 (09); 2017: 122-133 127

determined by interpolating data from an appropriate curve or by a
non-linear regression of the data by using different models (Chen
et al., 2013). Various models can be used to determine EC50. In
this work, we fit the experimental data to the logistic, Boltzmann
(sigmoidal), log (agonist) vs. normalized responsevariable slope
(doseresponse), and asymmetric sigmoidal (five parameters, 5P)
to predict EC50.
Figure 2 shows the effect of different concentration of
honey, bee pollen and propolis in free radical scavenging tests: (a)
DPPH assay and (b) ABTS assay. The data was fitted with
sigmoidal model, as an example. The results showed that the
relationship between radical inhibition and logarithm of the bee
products concentration is not a straight line, but a sigmoidal or
Sshape. Four mathematical model including logistic, sigmoidal,
dose-response and 5P were selected to fit the curve and estimated
EC50 of the bee products. The results indicated that these four
mathematic models could be used to fit our antioxidant data sets
and provide the EC50 values. The EC50 values of the same sample
among the four models did not show large difference (Table 2).
This might be because of the log-logistic based equation they used.
For the DPPH-assay, no statistical differences were found between
the EC50 of each model and the right-angled triangle method
(simple method) for honey (P>0.05). For bee pollen, the dose-
response and 5P models show no significant different between
their EC50 to that of the simple method (P>0.05) while the logistic
and sigmoidal models show opposite results (P0.05). Significant
differences were found between the EC50values of propolis
estimated by the four models to the simple method (P0.05).
However, the EC50 value obtained from the 5P
model was the closest to the simple method. The ABTS-assay
estimated EC50 of the bee products is also represented in Table
2.The results of honey for the ABTS assay are similar to those for
the DPPH assay (P>0.05). For bee pollen, there are no statistic
significant between the EC50 from dose-respond and the 5P model
to that of the simple method (P>0.05). For propolis, only dose-
response model shows no significant difference between its EC50to
that of simple method (P>0.05). These results indicated that it
might be better to use dose-response and 5P models for prediction
of the EC50 of the bee products via DPPH assay and to use dose-
response via ABTS assay. The reason that the 5P model was more
appropriate than the logistic and sigmoidal models in estimation of
EC50 for DPPH assay might be an impact of number of its
parameters in the log–logistic model. Dose-response was more
appropriate to estimate the EC50 than the other models for ABTS
assays might be because the normalizedresponses were used in an
equation. Dose-response, a log–logistic model, has four-parameter
as logistic and sigmoidal models but the response is normalized to
run from 0% to 100%. This model assumes that the data have been
normalized thus forces the curve to run between 0100%. Then the
EC50 is reflected as a response equal to 50%. Nonlinear modeling
with data normalization and constrains has been found to produce
more sigmoidal curves than nonlinear modeling without data
manipulation (Wenner et al., 2011). Wenner et al. (2011) have
reported that normalizing and constraining parameters increased
statistical power and minimized the need to exclude data because
of poor curve fitting. Although the overall interpretation of the two
modeling; with and without normalization methods of curve fitting
was similar.

Table 2: Estimated EC50 (mg/mL) of honey, bee pollen and propolis obtained by the different models: above, using DPPH assay; below: using ABTS assay.
DPPH assay
sample right-angled triangle logistic sigmoidal doseresponse 5P
α-tocopherol 0.055 ± 0.001 a, A 0.067 ± 0.004 a, B 0.068 ± 0.004 a, B 0.051 ± 0.001 a, A 0.060 ± 0.002 a, C
ascorbic acid 0.023 ± 0.002 b, AB 0.025 ± 0.001 b, B 0.026 ± 0.001 b, B 0.021 ± 0.002 b, A 0.024 ± 0.001 b, B
honey 276.7 ± 6.117 c, A 361.4 ± 96.86 c, A 317.4 ± 78.52 c, A 267.3 ± 15.99 c, A 283.8 ± 75.90 c, A
bee pollen 1.499 ± 0.015 d, AB 2.376 ± 0.047 d, C 1.920 ± 0.031 d, D 1.396 ± 0.021 d, A 1.625 ± 0.224 d, B
e, A e, B e, C e, D
propolis 0.150 ± 0.004 0.171 ± 0.004 0.180 ± 0.003 0.136 ± 0.005 0.161 ± 0.004 e, E
ABTS assay
sample right-angled triangle logistic sigmoidal doseresponse 5P
α-tocopherol 0.032 ± 0.002 a, ABC 0.036 ± 0.002 a, BC 0.037 ± 0.001 a, C 0.032 ± 0.003 a, AB 0.028 ± 0.004 a, A
ascorbic acid 0.023 ± 0.002 b, A 0.023 ± 0.004 b, A 0.023 ± 0.004 b, A 0.022 ± 0.002 b, A 0.022 ± 0.004 a, A
c, A c, A c, A c, A
honey 92.29 ± 5.638 93.41 ± 2.407 92.20 ± 2.174 95.02 ± 9.776 81.61 ± 10.34 b, A
bee pollen 0.560 ± 0.039 d, A 0.744 ± 0.117 d, B 0.744 ± 0.117 d, B 0.566 ± 0.024 d, A 0.698 ± 0.088 c, AB
propolis 0.054 ± 0.008 e, A 0.068 ± 0.006 e, B 0.073 ± 0.002 e, B 0.054 ± 0.009 e, A 0.073 ± 0.004 d, B
Note: Values followed by the same lowercase letter within the same column and for the same uppercase letter within the same row are not statistically significantly
different (p0.05).
128 Suriyatem et al. / Journal of Applied Pharmaceutical Science 7 (09); 2017: 122-133

Fig. 2: Effect of different concentration of honey, bee pollen and propolis in free radical scavenging tests: (a) DPPH assay and (b) ABTS assay. Data are fitted
with sigmoidal model.

Antimicrobial ability particularly dangerous pathogen due to its resistance to many

S. aureus is a gram-positive bacterium, which is an
important cause of gastroenteritis resulting from the consumption
of contaminated food (Loir et al., 2003). B. cereus is a gram-
positive bacterium, common soil saprophyte and is easily spread to
many types of foods (Granum Lund, 1997). E. coli is a gram-
negative bacterium that can be found in contaminated water or
food, especially raw vegetables and raw meat products
(Siripatrawan et al., 2013). It has been identified as a
commonly used antibiotics (Rahman et al., 2010). These three
bacteria are commonly recognized to cause food poisoning or food
spoilage.
The MIC values of Thai honey, bee pollen and propolis
against S. aureus, B. cereus and E. coli are listed in Table 3.A
turbidity assay was used to identify the growth of microorganisms
compared to the positive control. Each bee product tested caused
inhibition of bacterial growth. The MIC values of honey were of
 Suriyatem et al. / Journal of Applied Pharmaceutical Science 7 (09); 2017: 122-133 129

340 mg/mL for S. aureus and B. cereus while it was680 mg/mL
For E. coli. Concentrations of 153.71–614.83 mg/mL of pollen
extract inhibit growth against these three microorganisms. The
MIC values of the propolis were 1.88, 0.94 and 3.75 mg/mL
against S. aureus, B. cereus and E.coli, respectively. The MBC
values of the three types of bee products against the
microorganisms grown in NA plate are listed in Table 3. The
digital photographs of the MBC determination for honey, bee
pollen and propolis against S. aureus, B. cereus and E. coli are
shown in Figures3, 4 and5 respectively. NA plates streaked
from positive control tubes show the appearance of colonies of
S. aureus (Figure 3d), B. cereus (Figure 4d) and E.coli (Figure 5d).
The MBC assays showed growth, no growth and inhibition of
growth following bacterial streaks (Figure 3-5). Honey was lethal
to B. cereus and E. coli at the same concentration (680 mg/mL),
but did not kill S. aureus. A concentration of 614.83 mg/mL of the
pollen extract demonstrated effectiveness in killing S. aureus
while only 307.42 mg/mL was sufficient to kill B. cereus and E.
coli. The MBC of the propolis extract was 15.01 mg/mL for all the
bacteria S. aureus, B. cereus and E. coli. It was found that the
propolis extract solution had the lowest MIC and MBC values for
these bacteria.

Table 3: MIC values (mg/mL) and MBC values (mg/mL) of Thai bee products against food borne microorganisms.
Abbreviations:N.D. – without efficacy.
Microorganism Honey Pollen Propolis
MIC MBC MIC MBC MIC MBC
S. aureus 517 340.0 N.D. 153.7 614.8 1.9 15.0
B. cereus 687 340.0 680.0 153.7 307.4 0.9 15.0
E. coli 1261 680.0 680.0 153.7 307.4 3.8 15.0

Fig. 3: Digital photographs of the MBC of (a) honey. (b) bee pollen and (c) propolis against S. aureus and (d) control.
130 Suriyatem et al. / Journal of Applied Pharmaceutical Science 7 (09); 2017: 122-133

Fig. 4: Digital photographs of the MBC of (a) honey. (b) bee pollen and (c) propolis against B. cereus. and (d) control.

The MIC and MBC techniques are simple and easily
used to investigate the inhibitory doses of antibiotics or
disinfectants for particular bacteria (Rahman et al., 2010). In this
work, honey, pollen and propolis demonstrated antibacterial
activity against S. aureus, B. cereus and E. coli via the MIC and
MBC techniques. However, honey did not show an MBC against
S. aureus. The antimicrobial activity of honey is due to its osmotic
effect, natural acidity, hydrogen peroxide, phenolic acids,
flavonoids and lysozyme (Boukraâ et al., 2013). The antimicrobial
activity of pollen is attributed to its phenolic compounds (Boukraâ
et al., 2013), and the antimicrobial activity of propolis is caused by
the phenolic compounds such as flavonoids (Boukraâ et al., 2013).
Different antimicrobial agents possess different mechanisms.
The mechanism of antimicrobial resistance of honey, bee pollen
and propolis is involved degrading the cytoplasm membrane of the
bacteria (BellikBoukraâ, 2012). This leads to a loss of potassium
ions and the damage effected provoking cell autolysis
(BellikBoukraâ, 2012). Quercetin, a flavonoid found in both honey
and propolis, can increase membrane permeability of the bacterial
and dissipates bacterial potency (Mirzoeva et al., 1997). This
makes the bacteria lose their motility, membrane transport and
capacity to synthesis adenosine triphosphate (ATP). The present
study revealed that these bee products seemed to inhibit the gram-
positive bacteria more than gram-negative. This is in an agreement
with Brazilian propolis studied by Schmidt et al. (2014). In
general, plant extracts normally have a higher activity against
 Suriyatem et al. / Journal of Applied Pharmaceutical Science 7 (09); 2017: 122-133
gram-positive bacteria than gram-negative bacteria (Rahman et al.,
2010). Gram-negative bacteria are more resistant than the gram-
positive bacteria because they have more complex chemical
structures(Morais et al., 2011). This bacterial group has a
polysaccharide as one of the components in the cell wall, which is
involved in the antigenicity, toxicity and pathogenicity of the
microorganisms. Furthermore, the gram-negative bacteria possess
a higher lipid amount than that observed in gram-positive bacteria
(Morais et al., 2011).This lipid is a component of an endotoxin,
which has responsible toxicity in the cell wall of gram-negative
bacteria.
Graikou et al. (2011) reported that the MIC values of a
Greek pollen-methanol extract were 0.74 and >10 mg/mL against
S. aureus and E. coli, respectively. Morais et al. (2011) found that
the honeybee-collected pollen from Portuguese Natural Parks
provided the MIC of 0.17% (w/v) for B. cereus, 0.21% (w/v) for S.
aureus, and <5% (w/v) for E. coli. Choi et al. (2006) verified that
the propolis from Korea had much more powerful antimicrobial
activity than another from Brazil. Rahman et al. (2010)
.
Fig. 5: Digital photographs of the MBC of (a) honey. (b) bee pollen and (c) propolis against E. coli, and (d) control.
131
investigated propolis and honey from Canada against S. aureus
and E. coli. Propolis and honey concentrations at rates of 2.74–
5.48 and 375.0 mg/mL could inhibit S. aureus. For E. coli,
negative growth was found in propolis at a concentration of only
5.48 mg/mL, but honey was no effective. These MIC and MBC
values are different than the MIC and the MBC found to be active
in this work. These variations of antimicrobial property of honey,
bee pollen and propolis may be because of the different plants in
the sources where the bee lives. In addition, the results showed
that the antimicrobial activity of bee products in this study related
to total phenol content of the extracts as shown in Table 1. This is
because the phenolic compounds are the main sources of
antimicrobial action of honey, bee pollen and propolis. The other
works in bee products by Choi et al. (2006) and Morais et al.
(2011) were also in agreement. Miorin et al. (2003) suggested that
the effectiveness of honey or propolis depends on differences in
chemical composition, bee species and geographic region. Another
factors such as the nature of the phenolic fraction might be
involved (Morais et al., 2011), and they should be further study.
132 Suriyatem et al. / Journal of Applied Pharmaceutical Science 7 (09); 2017: 122-133
CONCLUSIONS
This work emphasized the value of honey, bee pollen and
propolis from Thailand as essential sources of natural antioxidant
and antimicrobial. Among these bee products, propolis possessed
the most powerful anti-free radical and antibacterial activities
following by bee pollen and honey, respectively. Phenolic
compounds play an important role to their strong effectiveness. In
order to evaluate the prediction of the EC50, four mathematic
equations logistic, sigmoidal, dose-response and 5P models could
be applied for calculations of EC50 values. Among these four
models, dose-response and 5P gave the nearest results to a right-
angled triangle method as a reference. Thus we recommended
dose-response and 5P model as the effective methods for the curve
fitting and prediction the EC50 via DPPH and ABTS assays. Future
work, we are interested to study the incorporating of the bee
products in the packaging materials in order to extend shelf life of
the prospective product.
ACKNOWLEDGEMENTS
The authors thank Assist. Prof. Dr. Chitsiti (Thongsorn)
Rachtanapan for her help.
Financial support and sponsorship: The authors are grateful to
the Thailand Research Fund through the Royal Golden Jubilee
Ph.D. Program (Grant No. PHD/0063/2555), the Graduate School,
Chiang Mai University and Faculty of Agro-Industry, Chiang Mai
University for financial support.
Conflict of Interests: There are no conflicts of interest.
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How to cite this article:
Suriyatem R, Auras RA, Intipunya P, Rachtanapun P. Predictive
mathematical modeling for EC50 calculation of antioxidant activity
and antibacterial ability of Thai bee products. J App Pharm Sci,
2017; 7 (09): 122-133.